Novel asymmetrical azines appending 1,3,4-thiadiazole sulfonamide: synthesis, molecular structure analyses, in silico ADME, and cytotoxic effect

Toward finding potential and novel anticancer agents, we designed and prepared novel differently substituted unsymmetrical azine-modified thiadiazole sulfonamide derivatives using the “combi-targeting approach”. An efficient procedure for synthesizing the designed compounds starts with 5-acetyl-3-N-(4-sulfamoylphenyl)-2-imino-1,3,4-thiadi-azoline 4. The E/Z configuration for compound 5 was investigated based on spectral analysis combined with quantum mechanical calculation applying the DFT-B3LYP method and 6-31G(d) basis set. The computational results found that the E isomer was energetically more favorable than the Z isomer by 2.21 kcal mol−1. Moreover, 1H and 13C chemical shifts for the E and Z isomers in DMSO were predicted using the GIAO-B3LYP/6-31G(d) computations and IEF-PCM solvation model. The computed chemical shifts for both isomers are consistent with those observed experimentally, indicating that they exist in the solution phase. Moreover, the E/Z configuration for the synthesized azines 7a–c, 9, 11, 13, 15a and 15b was also studied theoretically using the DFT-B3LYP/6-31G(d) calculations. In silico prediction for the biological activities was reported regarding the HOMO–LUMO energy gaps and molecular reactivity descriptors besides the ADMT/drug-likeness properties. The cytotoxic effect of the synthesized compounds has been assayed via the determination of their IC50.


Introduction
Current obstacles to treating cancer include the emergence of drug resistance and unfavorable off-target effects of anticancer drugs, which energizes medicinal chemists to continuously produce novel anticancer medications with high efficacy and low toxicity. 1,2 One of the most promising candidates in the eld of synthetic drugs is sulphonamides (Fig. 1a). [3][4][5][6] The thiadiazole platform constitutes intriguing and rapidly expanding sulfonamide derivative systems. Several medicines on the market are related to this system, making it a exible tool for drug design (Fig. 1b). 7,8 According to a literature survey, azines and derivatives serve as crucial structural components in various versatile scaffolds with a wide range of drug applications (Fig. 1c). 9 Azines have recently gained attention for congurations, and tautomers that profoundly affect biochemical processes. 10 Recently, a vital approach/strategy in drug discovery is the amalgamation of two or more complete medications into a single molecular structure, known as a combi-molecule, which may be a good solution to achieve bioactive molecules, with high potency and different mechanisms of action, due to the synergistic effect. 11 Hence, cohesive systems incorporating 1,3,4-thiadiazole sulfonamide with azines may help design new anticancer hybrids to improve biological properties.
To investigate the application of a combi-molecule strategy, we designed and prepared novel compounds 7a-c, 9,11,13 and 15a-b in this work by linking azines fragments and thiadiazole sulfonamides. These compounds were then subjected to cytotoxic assays on three cancerous cell lines. Their cytotoxic assay was comparable to the positive control staurosporine in the low micromolar region. Moreover, we employ quantum mechanical (QM) computations to provide theoretical analyses for compound 5, including the E/Z congurations and conformational study toward free rotatable single bonds. Thus, QM calculations were reported using the density functional theory (DFT) at the level of B3LYP 12 and 6-31G(d) basis set. Owing to its reliable accuracy and reduced computation time, the B3LYP/6-31G(d) calculation was widely recognized and applied for theoretical studies of organic molecules of medium-large size. 13,14 The 1 H and 13 C NMR chemical shis (d, ppm) were also computed by applying the approach of gauge-invariant atomic orbitals, GIAOs,15 to investigate the E/Z conguration of 5 in the solution phase. In silico techniques have been widely applied to drug screening. [16][17][18][19] Various computational tools and methods may be used to identify the candidate drug from other compounds depending on multiple features such as physicochemical/pharmacokinetic parameters and drug-likeness. Herein, QM calculations were carried out for the synthesized compounds to correlate their structures with biological activities via analysis of the FMOs and quantum chemical descriptors. To assess the synthesized compounds 7a-c, 9, 11, 13, 15a and 15b as drug candidates, the SwissADME 20 and pkCSM 21 servers were used to predict physicochemical characteristics, drug-likeness, and ADMET properties.

Chemistry
To synthesize the target compounds 7a-c, 9, 11, 13, 15a and 15b the synthetic sequence starts with the preparation of 5-acetyl-3-N-(4-sulfamoylphenyl)-2-imino-1, 3,4-thiadiazoline 4 as commencing material. 1,3,4-Thiadiazoline 4 was efficiently prepared via a cyclization reaction of freshly synthesized 2-oxo-N-(4-sulfamoylphenyl)propanehydrazonoyl chloride 3, obtained from the Japp-Klingemann reaction of 3-chloro-2,4pentanedione with diazonium chloride of sulfanilamide 2 in a buffered ethanolic solution, with an aqueous ethanolic solution of ammonium thiocyanate under reux (Scheme 1). Compound 4's structure was determined via microanalysis and spectral data. In the IR spectrum, three bands were observed at 3352, 3289, and 3277 cm −1 , indicating the existence of primary and secondary N-H stretching vibration, respectively. The bands at 1692, 1330, and 1298 cm −1 indicated the existence of a carbonyl group (C]O) and sulfonamide group (SO 2 NH 2 ), respectively. The 1 H-NMR spectrum displayed three singlet signals at d 2.50, 7.45, and 9.62 ppm, characteristic for CH 3 , SONH 2 , and NH protons, respectively, as well as two doublet signals, resonating at d 7.92 and 8.18 ppm with the identical coupling constant value (J = 9.35 Hz) and integrating for four protons indicating the existence of 4-disubstituted benzene. Its 13 C-NMR spectrum showed the presence of eight signals which agrees with its molecular structure. The signals of CH 3 and a carbonyl carbon resonate at 24.88 and 189.83 ppm, respectively. In the mass spectrum (MS), the molecular ion peak (M + ) for 4 was found at m/z = 298, which is compatible with its molecular formula (C 10 Condensation of 5-acetyl-3-N-(4-sulfamoylphenyl)-2-imino-1,3,4-thiadiazoline 4 with hydrazine afford the respective 4-(5-(1-hydrazonoethyl)-2-imino-1,3,4-thiadiazol-3(2H)-yl)benzenesulfonamide 5 (Scheme 1). The structure of later hydrazone 5 was conrmed through spectroscopic analyses. An examination of its IR spectrum revealed the lack of a carbonyl absorption band and the existence of azomethine (C]N) and amino groups at wavenumbers 1645, 3416, and 3285 cm −1 . Interestingly, the 1 H-NMR spectrum of hydrazone 5 showed two sets of resonances that supported the presence of 5 in two isomeric forms. The separation of signals of aromatic, -NH 2 , and CH 3 protons in the two isomers is well resolved. The population ratio of the major and minor isomers is (52 : 48). Based on previous studies, the most stable and the major isomer is assigned to the Eisomer around the C]N bond. 22 That is supported by the observed chemical shi value of the hydrazone-NH 2 protons in E-and Z-isomers. In E-isomer, the hydrazone-NH 2 protons appear as a singlet signal at d 5.66 ppm. In comparison, Zisomer resonates as two separate singlet signals at 7.33 and 7.32 ppm due to the possible formation of intramolecular Hbonds between N-H proton and C]N group in the thiadiazole ring as shown in Fig. 2. According to MS, the M + at m/z is the molecular weight.

Quantum mechanical study
2.2.1. Molecular structure analyses. Initially, the Z/E congurations were theoretically investigated for 5 according to the orientation of amino N and thiadiazol C attached to the C]N bond where they are in the same (Z isomer) or opposite sides (E isomer), see Fig. 3. The computational outcomes using B3LYP functional combined with the 6-31G(d) basis set reveal that the E conguration is more stable than the Z form by 769 cm −1 (2.21 kcal mol −1 ). While adopting the E/Z congurations, we need to explore the exact conformation of -C(CH 3 ) NNH 2 , phenyl sulfonamide, sulfonamide, and methyl moieties which result from free rotation about C 2 -C 6 , N 4 -C 15 , C 18 -S 25 and C 6 -C 7 single bonds, respectively. For this purpose, a relaxed scan of the PES was performed throughout the rotation of the dihedral angles s N 8 C 6 C 2 N 3 , s C 16 C 15 N 4 N 3 , s N 28 S 25 C 18 C 19, and s H 12 C 7 C 6 C 2 from 0°to 360°in steps of 10°proceeded by optimization process aer every scan point. The obtained curves of the PES scan using B3LYP/6-31G(d) calculations for E and Z isomers are given at ESI ( Fig. S1 and S2 †). For the E isomer, the predicted PES curve from the rotation of -C(CH 3 )NNH 2 moiety shows a global minimum when s N 8 C 6 C 2 N 3 reaches 180.0°in which the imine bonds (C]N) are trans to each other.
In contrast, minimum energy for Z-isomer was obtained at s N 8 C 6 C 2 N 3 equal to 0.0°where both C]N bonds are cis to each other where stabilization could be attributed to intra-molecular H-bonding interaction between amino hydrogen and thiadiazol nitrogen. Moreover, the energetically favored conformation of benzenesulfonamide moiety (-C 6 H 4 SO 2 NH 2 ) concerning the thiadiazol ring was assigned at s C 16 C 15 N 4 N 3 of 10.0°where the phenyl ring is almost planar towards the thiadiazol ring. The predicted curve for the scan of PES throughout the rotation of sulfonamide moiety for both E and Z isomer exhibit a minimum conformation at s N 28 S 25 C 18 C 19 equal to 260.0°in which the amino group is perpendicular to the phenyl ring. The internal rotation of the methyl group gives rise to a minimum at s H 12 C 7 C 6 C 2 equal to 60.0°and 0.0°for Z and E conguration, where the methyl group is orientated in eclipsed and staggered conformation to the adjacent C]N bond, respectively. In conclusion, opposite conformations of C(CH 3 )NNH 2 and methyl moieties were obtained for E and Z isomers. In contrast to the E isomer, the global minimum of the Z isomer has the C]N bond in cis orientation to the thiadiazol C]N bond with the eclipsed conformation of the methyl group towards the C]N bond (Fig. 3).
The measured 13 C NMR spectrum for 5 displays 20 signals, double the number of carbon atoms, and reveals the presence of both Z and E isomers in the solution phase. Thus, the 1 H and 13 C NMR chemical shis (d in ppm) were calculated for both isomers using B3LYP/6-31G(d) calculations and compared to those observed experimentally. The computed 1 H and 13 C chemical shis for Z and E isomers have equivalent values in the experimental spectra, as Table 1 shows that both isomers exist in the solution phase. In contrast to the E isomer, one proton of amino-hydrazone moiety (H 11 ) for the Z isomer was predicted to resonate at 7.31 ppm and matches the observed signal at 7.33 ppm, conrming the presence of the Z isomer in the solution phase. The downeld shi for H 11 accounts for the intra-molecular H bonding with adjacent thiadiazol nitrogen. The 1 H NMR spectrum shows three signals at 7. 36, 7.36, and 7.43 ppm corresponding to amino protons of sulfonamide moiety (H 29 and H 30 ) in excellent agreement with those calculated for both E and Z isomers, 7.34-7.41 ppm. It's worth noting that, compared to tetramethylsilane (TMS), the prediction of chemical shis for amino protons was improved when a multistandard technique 24,25 was applied using a comparable skeleton as a reference.
The predicted chemical shi for the Z isomer at 126.49 ppm belongs to the hydrazone carbon atom (C 6 ) and better matches the 13 C signal observed at 126.80 ppm. The counterpart value for the E isomer was computed at 138.02 ppm, which is consistent with the signal observed at 139.07 ppm. The experimental 13 C NMR spectrum shows signals at 151. 26   The E/Z conguration for the synthesized products 7a-c, 9,11,13 and 15a-b were theoretically explored based on the orientation around the hydrazonyl C]N bonds, which results in four possible congurations, 1 (EE), 2 (ZZ), 3 (EZ) and 4 (ZE). Therefore, a full geometry optimization was carried out for each conguration followed by frequency applying B3LYP method at 6-31G(d) basis set. The ESI † provides the equilibrium geometries and computed energy difference for the suggested congurations (Fig. S3-S10/Table S1 †). For all synthesized compounds, the computational outcomes revealed EE (1) isomer to be the favored conguration with the lowest energy. Fig. 4 shows the predicted equilibrium geometries for the most stable conguration (1, EE) for 7a-c, 9, 11, 13, 15a and 15b.
The impact of FMOs and their associated molecular reactivity descriptors on molecule biological reactivity has recently been considered. 26,27 Herein, The FMOs were predicted for the optimized geometries of the synthesized molecules (5, 7a-c, 9,11,13 and 15a-b) to evaluate the reactivity and to correlate their biological activities. Subsequently, the computed energies for HOMO and LUMO were used to calculate a set of quantum chemical descriptors (Table 2), which are useful in assessing the molecule's overall reactivity. 28,29 The distribution for electron density in HOMOs and LUMOs for the investigated compounds is shown in Fig. 5, along with their energy gaps predicted using the B3LYP/6-31G(d) computations. The HOMO is largely localized on the thiadiazol ring and phenyl ring of the benzenesulfonamide moiety except for 7b, where the HOMO is mostly distributed over the dimethylphenylamine moiety. The LUMO localized over the whole molecule for all synthesized molecules except the region containing the benzenesulfonamide moiety.
The energy gap (E HOMO -E LUMO ) is a valuable sign of a molecule's chemical reactivity and kinetic stability. A molecule with a small energy gap is more polarized and has a higher chemical reactivity and lower kinetic stability. 30 Furthermore, Small Fig. 4 Optimized geometries of 7a-c, 9,11,13 and 15a-b obtained from B3LYP/6-31G(d) calculations. energy gaps suggest that the molecule undergoes a large intramolecular charge transfer, which might affect the molecule's biological activity. 31 The energy gaps calculated for the synthesized compounds ranged from 2.96 to 4.11 eV, comparable to the reported values for bioactive molecules [32][33][34] and that computed for the standard reference drug, staurosporine (4.12 eV). For all compounds, the calculated energy gap E g ( Table 2) decreases in the order 5 (4.11 eV) > 9 > 7a > 11 > 15a > 15b = 7b > 13 > 7c (2.96 eV). These results are compatible with the observed high activities for 7c, 13, 15b, and 15a against HepG-2 and Caco2 (Table 3).
Chemical potential (m) is the inverse of electronegativity (c), and it denes how much energy a molecule absorbs or releases during a chemical process. Both descriptors signicantly impact a molecule's inhibitory effectiveness. 35 Compound 7c, with a high c value (of 4.69 eV), is more active than 7a and 7b with low values (4.03 and 3.74 eV), which is attributed to the presence of the nitro group in 7c and validates the observed higher activity against MCF-7 (Table 3). Also, the hardness value for 7c (1.48 eV) is lower than that calculated for 7a and 7b. It shows that it is a soer and more reactive molecule compared to 7a and 7b, in agreement with the estimated energy gap, see Table 2. Owing to the values of the electrophilicity index (u), organic compounds were categorized as strong (u > 1.5 eV), moderate (0.8 < u < 1.5 eV), or weak (u < 0.8 eV) electrophiles. 36 Compounds 7c and 13 are powerful electrophiles in this study, with u values of 7.43 and 6.17, respectively. Compound 9 has the lowest biological activity against Caco2, which might be explained by its high energy gap (3.49 eV) and poor electrophilicity (4.01 eV) between all the synthesized compounds. To support the combi-targeting technique in this study, QM descriptors were computed for three commercial drugs with similar cores, valdecoxib, methazolamide, and ZSL4 (Fig. 1, Table 2). As a result, the computed E g and h values for the ZSL4 drug matched those predicted for compound 5 and staurosporine. Also, compounds 7a and 11 have m values of −4.03 and −3.97 eV, respectively, and are similar to those obtained for valdecoxib and ZSL4 drugs.
The dipole moment is also considered when correlating the molecule's biological activity, which might inuence the degree of interaction between drugs and the active sites of protein. 37 Compounds 15a-b, for example, are more active against Caco2 and MCF-7 than 13, which may be explained by the fact that 15a-b has a higher total dipole moment (8.37-8.77 Debye) than 13 (5.25). As shown in Table 2, compounds 7b and 9 have high total dipole moments (12.07 and 11.91 Debye, respectively) and exhibit high polarizabilities (407.38 and 402.22 a.u.), indicating that they are a good candidate for non-linear optical (NLO) materials. 38 2.2.2. ADMET prediction. Besides the high potency, the drug candidate's success involves favorable ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties. 39 The ADMET prediction models have been introduced as an additional tool to aid drug discovery. 16 This study predicted the in silico ADMET characteristics of all synthesized compounds 7a-c, 9, 11, 13, 15a and 15b using the SwissADME 20 and pkCSM 21 servers, as shown in Table 4. The synthesized compounds show percent absorption ranged from 73.44 to 78.33%, suggesting they are well absorbed via the human intestine. Table 4 shows that three compounds 7a-c have no violations of Lipinski's rule of ve 40 for drug-likeness features and are considered orally active drugs. Other compounds have just one violation. All the synthesized compounds 7a-c, 9, 11, 13, 15a and 15b were found to be AMES nontoxic in nature and exhibit lethal doses (LD 50 ) ranging from 2.02 to 3.32 mol kg −1 , indicating these compounds seem to be suitably safe.
The oral bioavailability was estimated using the Swis-sADME's bioavailability radar, which considers six physicochemical parameters: size, solubility, lipophilicity, polarity, exibility, and saturation. The bioavailable radars for the investigated compounds in this work are given (Fig. S11 at ESI †). Accordingly, the pink zone for all examined compounds has four parameters that provide physicochemical space  acceptable for oral bioavailability. However, saturation and polarity were found beyond the bioavailability radar's pink zone due to a low saturation (fraction of Csp 3 < 0.25) and high polarity (TPSA > 130 Å 2 ).

Computational details
Gaussian 09 package soware 43 was used to carry out all quantum chemical calculations using the method of DFT-B3LYP 12,13 combined with a standard 6-31G(d) basis set. The suggested geometries for the synthesized compounds were initially optimized using Pulay's gradient approach. 44 Frequency calculations were performed to verify that the optimized geometries are actual minimums with real wavenumbers. The geometry of the E and Z forms of 5 were reoptimized in DMSO solution using the IEF-PCM solvation model, 45 followed by 1 H and 13 C NMR chemical shi calculations applying the GIAO approach. 14,15 The multi-standard approach 24,25 was applied to get the theoretical chemical shis for E and Z isomers using the isotropic magnetic shielding values (s i , ppm) for H and C atoms acquired from the Gaussian output le. In this approach, methanol and benzene were used as references to predict sp 3 and sp 2 hybridized Cs/C-Hs, respectively, whereas comparable skeletons were used in the case of N-H protons. Thus, benzenesulfonamide, 46 acetophenonehydrazone, 47 and 5-iso-propyl-3methyl-2-imino-1,3,4-thiadiazole 48 were used as references to calculate the N-H chemical shis in the benzenesulfonamide, hydrazone and iminothiadiazoles moieties, respectively. Furthermore, the HOMO/LUMO (FMOs) and energy gaps were predicted for the optimized geometries of the synthesized compounds 7a-c, 9, 11, 13, 15a and 15b. Accordingly, molecular reactivity descriptors such as ionization potential, electron affinity, hardness, chemical potential, and electrophilicity index were calculated using the values of energies for FMOs. 28,29 The synthesized compounds' pharmacokinetics, drug-like characteristics, and toxicities were evaluated using the SwissADME 20 and pkCSM 21 online servers.

Biological evaluation
3.3.1. Cytotoxicity evaluation. HepG2, MCF-7, WI 38, Caco-2 cancer cell lines were cultured in complete media of RPMI and DMEM, respectively, at 5% carbon dioxide and 37°C following standard tissue culture work. The cells were grown in "10% fetal bovine serum (FBS) and 1% penicillin-streptomycin" in 96multiwell plate. All the synthesized compounds were screened for their cytotoxicity using 20 mL of MTT solution (Promega, USA) for 48 h using untreated and treated cells with concentrations of (0.01, 0.1, 1, 10, and 100 mM) for 48 h. 41,42 The plate was cultured for 3 hours. The percentage of cell viability was calculated following this equation: (100 − (A sample )/(A control )) × 100. An ELISA microplate reader was used to measure the absorbance at 690 nm to calculate the viability versus concentration, and the IC 50 value using GraphPad prism soware.

Conclusions
Herein, the combi-molecule strategy was used to synthesize a bundle of differently hybrid 1,3,4-thiadiazole sulfonamide derivatives and unsymmetrical azines 7a-c, 9, 11, 13, 15a and 15b started with 5-acetyl-3-N-(4-sulfamoylphenyl)-2-imino-1,3,4thiadiazoline 4. In the solution phase, compound 5 exists as a mixture of E and Z congurations, according to the results of computed/observed 1 H and 13 C NMR chemical shis. The DFT-B3LYP calculations favor the nal synthesized products in the EE conguration with reference to the azomethine C]N bonds. For the newly synthesized compounds, quantum chemical descriptors and drug-likeness properties were predicted and correlated to their in vitro bioactivities. Experimentally, the synthesized compounds were investigated for their anticancer effects in vitro against cancer cell lines: HepG-2, Caco-2, MCF-7, and WI-38, where they exhibited promising results.

Conflicts of interest
The authors declare that there are no conicts of interest.